טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGottlieb Yehonatan
SubjectResponse of Macrophages to Erythtrophagocytosis
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Dr. Esther Meyron Holtz
Full Thesis textFull thesis text - English Version


Abstract

Background: Erythrophagocytosis (EPC) is the process of removal of aged or damaged red blood cells (RBCs) by macrophages (MPs), mostly in the spleen. EPC is part of the systemic iron cycle as iron is released from degrading RBC-heme and is recycled to newly developing RBCs in the bone marrow. EPC poses a great metabolic stress for MPs when toxic materials are released from degraded RBCs.  Heme is degraded mainly by Heme oxygenase 1 (HO-1), into iron, CO and biliverdin. Senescence markers of RBCs that might trigger EPC have been studied extensively and EPC has been mimicked mainly by using manipulated erythrocytes in which senescence signals were induced by chemical or biological means. Heme degradation was also explored, but the exact subcellular site of heme degradation was never solved and the enzyme orientation on the ER membrane in vivo has not been elucidated.

Design and methods: The study was conducted in two parallel paths. The first path was to establish a model of EPC of physiologically aged RBCs. Blood, enriched with senescent erythrocytes was generated in vivo with a hypertransfusion (Ht) protocol. Senescence markers were evaluated in physiologically aged RBCs and compared to control senescent RBCs. The RBC population enriched with senescent cells was then used in an EPC-assays in vivo and in vitro. The second path was focused on heme degradation inside the MPs. Orientation of HOs in MPs before and after EPC was evaluated by fused constructs of HO-1 and HO-2 with fluorescent proteins. We also tried to evaluate the localization of HO-1 after EPC using an in vitro EPC model, followed by immunofluorescent labeling.

Results and conclusions: The large cohort of senescent erythrocytes in the RBC population from ht-mice carried ageing signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from ht-mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. But neither RBCs from ht-mice, nor from control mice were phagocytosed in vitro by primary MP cultures. HOs active site was determined to be on the cytosolic side of the ER membrane. We have also found that HO-1 is found in closed vicinity to the phagosome. The inefficient EPC in vitro emphasize the great difference between in vivo aged and in vitro manipulated RBCs and the difference between in vivo and in vitro processes.